Figure 4.

Defective ALC1-mediated nucleosome remodeling confers synthetic lethality with HRD

(A–E) Loss of ALC1 is synthetic lethal with HRD and leads to PARPi hypersensitivity. (A) Immunoblot of WCEs from DLD-1 WT and BRCA2^−/− cells following transduction with LentiCRISPR NT sgRNA and ALC1 sgRNA and clonal selection (no BRCA2/ALC1 double knockouts were recovered), probed with BRCA2 and ALC1. α-tubulin was used as a loading control. (B) Olaparib colony survival in DLD-1 BRCA2^+/+ALC1^+/+, BRCA2^−/−ALC1^−/−, BRCA2^−/−ALC1^+/+, and BRCA2^−/−ALC1^{Low expression}. Data are mean ± SEM normalized to untreated cells (n = 3 independent biological experiments). Solid lines show a nonlinear least-squares fit to a four-parameter dose-response model. (C) Survival in ALC1^+/+ and ALC1^−/− eHAP cells transfected with BRCA1-targeting short interfering RNAs (siRNAs). Cell survival was measured using CellTiter-Glo. Data are mean ± SEM normalized to ALC1^+/+ cells (n = 3 independent biological experiments). (D) Survival in ALC1^+/+ and ALC1^−/− eHAP cells transfected with BRCA2-targeting siRNAs. Cell survival was measured using CellTiter-Glo. Data are mean ± SEM normalized to ALC1^+/+ cells (n = 3 independent biological experiments). (E) Olaparib survival in ALC1^+/+ and ALC1^−/− eHAP transfected with non-targeting or BRCA2-targeting siRNAs. Data are mean ± SEM normalized to untreated cells (n = 3 independent biological experiments). Solid lines show a nonlinear least-squares fit to a four-parameter dose-response model.

(F) Quantification of a crystal violet proliferation assay in parental and ALC1-deleted BARD1^AID/AID cells ± IAA. Data are mean ± SD (n = 3 independent biological experiments).

(G) Quantification of a crystal violet proliferation assay in parental and ALC1-deleted 53BP1^−/−BARD1^AID/AID cells ± IAA. Data are mean ± SD (n = 3 independent biological experiments).

(H) ALC1^+/+ and ALC1^−/− eHAP cells transduced with indicated ALC1 constructs were transfected with non-targeting, BRCA1-targeting, or BRCA2-targeting siRNAs. Cell survival was measured using CellTiter-Glo. Data are mean ± SEM normalized to ALC1^+/+ cells for each siRNA (n = 3 independent biological experiments).

(I) Immunoblot of WCEs in ALC1^+/+ and ALC1^−/− iCAS9 eHAP cells transduced with ATM sgRNA following 72 h Dox, probed with antibodies against ALC1 and ATM. α-tubulin is used as a loading control.

(J) Representative images (n = 3 biologically independent experiments) of clonogenic survival assays in ALC1^+/+ and ALC1^−/− iCAS9 cells expressing NT and ATM sgRNA following 72 h Dox ± 250 nM Olaparib.

(K) Quantification of clonogenic survival assays in ALC1^+/+ and ALC1^−/− iCAS9 cells expressing NT sgRNA and ATM sgRNA following 72 h Dox ± 250 nM Olaparib. Data are mean ± SEM normalized to non-treated ALC1^+/+ NT sgRNA (n = 3 biologically independent experiments).

(L) Olaparib survival of ALC1^+/+ and ALC1^−/− iCAS9 cells transduced with NT sgRNA and ATM sgRNA following 72 h Dox. Data are mean ± SEM normalized to untreated cells (n = 3 independent biological experiments). Solid lines show a nonlinear least-squares fit to a four-parameter dose-response model.

(M) Hazard ratio analysis of breast cancer patients from TGCA according to ALC1 and BRCA2 expression.

(N) KM survival analysis of BRCA2^low breast cancer patients from TGCA according to ALC1 expression. ns, p > 0.05; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.